Determination And Biological Function Reasearch Of FabA And FabB In Xanthomonas Campestris Pv.Campestris

Xanthomonas campestris pv.campestris（Xcc）,is the main pathogenic bacterium of the cruciferous black rot,which causes worldwide economic crop damage and huge economic losses.The Fatty acid in Xcc includes saturated fatty acid,branched chain fatty acid and unsaturated fatty acid,while the unsaturated fatty acid synthesis pathway is still not be revealed,so research clearly of Xcc unsaturated fatty acid synthesis mechanism will provide the theoretical basis to find suitable antibacterial target.The unsaturated fatty acid synthesis with the FabA-FabB pathway in the type strain E.coli.Bioinformatics analysis shows that,there are two genes annotated for 3-hydroxy fatty acyl ACP dehydration isomerase and 3-ketone fatty acyl ACP synthetaseⅠ,XC₃651 and XC₃652,closely arranged with some overlap,shared a promoter sequence.However,they are not in fatty acid synthesis related gene cluster.XC₃651 has 74.2% similarity with EcFabA protein sequence,has the same conservative sites and the same two alpha helix structure with EcfabA;XC₃652 has 62.7% similarity with EcFab B,and has the same catalytic active center with EcFabB.We constructed pCC3（pBAD-XC₃651）and pCC4（pBAD-XC₃652）plasmids,then they were transformed into fabA temperature sensitive mutant strain of E.coli CY57 and fab B temperature sensitive mutant strain of E.coli CY242 separately.Complementary results showed that they restore CY57 and C Y242 growth in 42℃ no-growth temperature.It gives the preliminary evidence that XC₃651 encodes 3-hydroxy fatty acyl ACP dehydration isomerase（FabA）,and XC₃652 encodes 3-ketone fatty acyl ACP synthaseⅠ（Fab B）.In order to further proof the functions of XcFab A and XcFab B,we constructed prokaryotic expression vector p ET28 b serial plasmids pCC5、pCC6,expressed and purified the XcFabA、XcFab B proteins.Reconstructing the reaction system of fatty acid synthesis in vitro,to detecte the activity of two proteins,and results showed that XcFabA are able to catalyze 3-hydroxy Decanoyl ACP dehydrate and isomerism to cis-3-Decyl acryloxyethyl ACP;XcFabB can catalyze Octanoyl ACP and Malony ACP synthesis to 3-ketoacyl Decanoyl ACP.It effectively illustrates that XcFab A possess the activity of 3-hydroxy fatty acyl ACP dehydration isomerase,EcFab B has the activity of 3-ketone fatty acyl ACP synthase.This study comprehensive explored and experiented the biological function of Xcfab A and Xcfab B in Xcc.We obtained the mutant strain CC7（XcΔ fabA）、CC8（XcΔ fabB）and constructed the complementary strain CC11（XcΔ fabA/XcfabA）、CC12（XΔ fabB/XcfabB）with the Knock-out technology,and detected the physiological effect among this different strain.It turn out that there are no different in the growth of the mutant strains and wide typle strain under the condition of rich medium NYG or minimum medium XO at 30℃ or 15℃.Analyzing the fatty acid composition of this different strains still shows no obviously difference under the condition of rich medium NYG or minimum medium XO at 30℃.Detecting the pathogenic factor extracellular enzyme and exopolysaccharides shows no difference between this different strains.The stress resistance detecting revealed the Xcfab B mutant has an apparent lower stress resistance to NaCl than the wide strain Xcc,orther strains showed no difference to PH,SDS and NaCl.At last,we tested the pathogenicity of this differnet strains to the leaf of radish belong the cruciferous plants,also the Xcfab B mutant strain shows obviously decrease.This study demonstrated XC₃651 possess the function of 3-hydroxy fatty acyl ACP dehydration isomerase,XC₃651 possess the function of 3-ketone fatty acyl ACP synthaseⅠin vitro.However,it shows that XcfabA、XcfabB gene seems no directly relation with the Xcc unsaturated fatty acid synthesis mechanism in vivo.Therefore,there must be a novel unsaturated fatty acid synthesis mechanism in Xcc.